Compound and application thereof

ABSTRACT

The present invention relates to a novel compound and application thereof in the inhibition of HBV gene expression. The structure of the compound comprises an interfering nucleic acid for inhibiting HBV gene expression, transition points, and delivery chains of the interfering nucleic acid. By means of the delivery chains, two or three N-acetylgalactosamines can be introduced to an antisense strand 3′ end of such siRNA, and two or one N-acetylgalactosamine can be correspondingly introduced to a sense strand 5′ end, the total number of the introduced N-acetylgalactosamines being four. In vitro and in vivo pharmacological experiments prove that such a novel compound can continuously and efficiently inhibit HBV gene expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/623,569, which was the National Stage of International ApplicationNo. PCT/CN2020/097732, filed Jun. 23, 2020; which claims the benefit ofChinese Application No. 201910576037.1, filed Jun. 28, 2019, and ChineseApplication No. 201911281389.0, filed Dec. 13, 2019; the disclosure ofeach of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a novel compound and its application inthe inhibition of HBV gene expression. The structure of the compoundcomprises an interfering nucleic acid for inhibiting HBV geneexpression, transition points and delivery chains of the interferingnucleic acid. By means of the delivery chains, such siRNA can beintroduced with two to three N-acetylgalactosamines at the 3′ end of theantisense strand, and correspondingly, two to one N-acetylgalactosamineat the 5′ end of the sense strand, with the total number of theintroduced N-acetylgalactosamines being four. Pharmacologicalexperiments on HepG2 cells and transgenic mice demonstrate that, thisnovel compound can continuously inhibit the expression of HbsAg andHBeAg of HBV and HBV DNA.

REFERENCE TO A SEQUENCE LISTING

The present specification is being filed with a Sequence Listing inComputer Readable Form (CRF), which is entitled413A001US02_SEQ_LIST_ST25.txt of 28,637 bytes in size and created Aug.29, 2022; the content of which is incorporated herein by reference inits entirety.

BACKGROUND ART RNAi

RNAi (RNA interference) was discovered in an antisense RNA inhibitionexperiment on Caenorhabditis elegans carried out by Andrew Z. Fire etal. in 1998, and this process was named as RNAi. This discovery wasrecognized by Science as one of the top ten scientific advances in 2001,and ranked the first on the list of the top ten scientific advances in2002. Since then, siRNA with the mechanism of RNAi has attracted muchattention as a potential genetic therapeutic drug. In 2006, Andrew Z.Fire and Craig C. Mello won the Nobel Prize for Physiology or Medicinefor their contribution in the study of RNAi mechanism. RNAi can betriggered by double stranded RNA (dsRNA) in many organisms, includinganimals, plants and fungi. In the process of RNAi, a long-chain dsRNA iscleaved or “diced” into small fragments of 21 to 25 nucleotides inlength by an endonuclease known as “Dicer”. These small fragments areknown as small interfering RNA (siRNA), in which the antisense strand(Guide strand) is loaded onto Argonaute protein (AGO2). AGO2 loadingoccurs in a RISC-loading complex, which is a ternary complex composed ofan Argonaute protein, a Dicer and a dsRNA binding protein (brieflyreferred as TRBP). In the process of loading, the sense strands(Passenger strand) are cleaved by AGO2 and discharged. Then, AGO2utilizes the antisense strands to bind to mRNAs containing completecomplementary sequences, and catalyzes the cleavage of these mRNAs, suchthat mRNAs are cleaved to lose their function of translation template,which in turn prevents the synthesis of related proteins. Aftercleavage, the cleaved mRNAs are released, and the RISC-loading complexloaded with the antisense strand was recycled into another round ofcleavage.

According to statistics, among disease-related proteins in human body,more than about 80% of proteins cannot be targeted by currentlyconventional small molecule drugs and biological macromolecules, so theybelong to undruggable proteins. Gene therapy, aiming to treat diseasesthrough gene expression, silencing and other functions, is regarded inthe industry as the third generation of therapeutic drugs followingsmall chemical molecule drugs and biological macromolecules. Suchtherapy realizes the treatment of diseases at the genetic level and isnot restricted by undruggable proteins. As the most mainstream type ingene therapy, RNAi technology treats diseases at mRNA level, with muchhigher efficiency compared with the treatment by small chemical moleculedrugs and biological macromolecules at protein level. By use of RNAitechnology, sequences for sense strands and antisense strands of siRNAwith high specificity and effective inhibition can be designed accordingto particular gene sequences, and these single-strand sequences may besynthesized in solid phase, and then the sense strands and the antisensestrands are hybridized following the principle of base pairing in aparticular annealing buffer into siRNA, which is finally delivered tocorresponding target points in vivo through a carrier system to degradethe targeted mRNA and destroy the function of the targeted mRNA astranslation template, thereby blocking the synthesis of correspondingproteins.

Delivery System of siRNA

siRNA is labile in blood and tissues and prone to be degraded bynucleases. To improve the stability of siRNA, the skeleton of siRNA canbe modified. However, these chemical modifications only provide limitedprotection from nuclease degradation and may eventually affect theactivity of siRNA. Therefore, a delivery system is further needed toensure that siRNA can cross cell membranes efficiently. Because siRNAhas a large molecular mass with a large amount of negative charges and ahigh solubility in water, they cannot cross the cell membranes to getinto the cells.

A liposome has a basic structure consisting of a hydrophilic nucleus anda phospholipid bilayer. Due to the similarity and high biocompatibilityof phospholipid bilayer to a biological membrane, liposome was once themost popular and widely used siRNA carrier. In liposome-mediated siRNAdelivery, siRNA is mainly encapsulated inside the liposome to protectthe siRNA from nuclease degradation, thus improving the efficiency ofsiRNA passing the cell membrane barriers and promoting the uptake ofcells. Liposome includes e.g., anionic liposomes, pH-sensitiveliposomes, immunoliposomes, fusogenic liposomes and cationic liposomesand the like. Although some progresses have been made, liposomesthemselves are prone to triggering an inflammatory response, so beforeadministration of a liposome, various antihistamine and hormone drugs,such as Cetirizine and dexamethasone, must be used so as to reduce acuteinflammatory responses that may occur. Therefore, liposomes are notsuitable for all therapeutic areas in practical clinical applications,especially diseases with long treatment cycles such as chronic hepatitisB, and the cumulative toxicity that may be generated from long-term useis a potential safety hazard.

Asialoglycoprotein Receptor (ASGPR)

The asialoglycoprotein receptor (ASGPR) in liver is a receptorspecifically expressed in liver cells, and a highly efficient endocyticreceptor. Because the secondary end of various glycoproteins exposed byenzymatic or acid hydrolysis of sialic acid under physiologicalconditions in vivo is a galactose residue, the sugar specifically boundby ASGPR is galactosyl, and ASGPR is also known as a galactose-specificreceptor. Monosaccharide and polysaccharide molecules, such asgalactose, galactosamine, N-acetylgalactosamine and the like, have ahigh affinity to ASGPR. The main physiological function of ASGPR is tomediate the removal of asialoglycoprotein, lipoprotein and othersubstances from the blood, and it is closely related to the occurrenceand development of viral hepatitis, liver cirrhosis, liver cancer andother liver diseases. The discovered property of ASGPR has an importanteffect on the diagnosis and treatment of liver-derived diseases (AshwellG, Harford J, Carbohydrate specific Receptors of the Liver, Ann RevBiochem 1982 51:531-554). Therapeutic drugs containing galactose orgalactosamine or derivatives thereof in their structures for treatingliver-derived diseases may have a specific affinity with ASGPR, so thatthey may have an active hepatic targeting property, without the need ofother carrier systems for delivery.

SUMMARY OF THE INVENTION

The present invention relates to a novel compound and an applicationthereof in the inhibition of HBV gene expression. The structure of thecompound comprises an interfering nucleic acid for inhibiting HBV geneexpression, transition points and delivery chains of the interferingnucleic acid. The delivery chains are linked to the interfering nucleicacid through transition points. By means of the delivery chains, suchsiRNA can be introduced with two or three N-acetylgalactosamines at the3′ end of the antisense strand, and correspondingly, two or oneN-acetylgalactosamine at 5′ end of the sense strand, with a total numberof N-acetylgalactosamines being four, which is a completely novelintroduction manner. In vitro and in vivo pharmacological experimentsdemonstrate that, this novel compound can continuously and efficientlyinhibit the expression of HBsAg, HBeAg of HBV and HBV DNA.

In one aspect, the present invention provides a compound comprising aninterfering nucleic acid for inhibiting HBV gene expression, transitionpoints and delivery chains of the interfering nucleic acid in itsstructure, wherein the delivery chains consist of a linking chain D, alinker B, a branched chain L and a liver targeting specific ligand X andare linked to the interfering nucleic acid through transition pointsR₁/R₂, wherein the compound has a structure of formula (I):

wherein:

-   -   when n is 1, m is 3; when n is 2, m is also 2;    -   R₁ is —NH(CH₂)_(x)CH₂—, wherein x may be an integer of 3-10;    -   R₂ is —NHCH₂CH(OH)CH₂—, or another nitrogen-containing structure        with both primary and secondary alcohol moieties or only primary        alcohol moieties, which may be a linear chain or a linear chain        with one or more branched chains, and may also be a cyclic        structure, preferably, R₂ may be a pyrrole or piperidine ring        with primary and secondary alcohol moieties, and specifically,        R₂ is selected from the following structures:

-   -   the liver targeting specific ligand X is selected from        galactose, galactosamine and N-acetylgalactosamine;    -   the branched chain L is a C3-C18 linear chain containing        carbonyl, amido, phosphoryl, oxygen atom or a combination of        these groups;    -   the linker B is selected from the following structural formulae:

-   -   wherein, A₁ is C, O, S or NH; r1 is a positive integer of 1-15,        r2 is an integer of 0-5; A₂ is C, O, S, amino, carbonyl, amido,        phosphoryl or thiophosphoryl;    -   the linking chain D contains 5 to 20 carbon atoms, and contains        amino, carbonyl, amido, oxygen atom, sulphur atom,        thiophosphoryl, phosphoryl, cyclic structure or a combination of        these groups.

In the above technical solution, the interfering nucleic acid includes,but not limited to, siRNA, miRNA and Agomir, preferably is a siRNA,further preferably is an anti-hepatitis B virus siRNA.

The sequence of the siRNA used for 19 mer of HBV RNAi is designedaccording to the target sequences of HBV cDNA (GenBankAccession#AF100309.1). These target sequences include 19 mer del core areas andcorresponding extended or shifted DNA sequences dominated by these coreareas. It intends to find optimal efficient sequences through basictarget sites, and part or all of these sequences can be suitable for thetarget sites, and can be applied to treatment of chronic hepatitis B.The 19 mer nucleotide sequence at the target site comprises two strands,a sense strand (S) and an antisense strand (AS), wherein, the 19^(th)nucleotide (5′→3′) on the sense strand can form a base pair with thefirst nucleotide (5′→3′) on the antisense strand according to theWatson-Crick principle. Following this principle, the 1^(st) to 19^(th)bases of the sense strand (5′→3′) and the 19^(th) to 1^(st) bases of theantisense strand (5′→3′) can form a double strand by pairing with theircorresponding bases. One to three unpaired bases can be allowed at theends of the double strand. In the present invention, HBV siRNA basicsequences can be screened according to practical applications. Through3′ or 5′ end displacement on the basis of the basic sequence, moreefficient and specific sequences can be screened. According to the studyresults on the siRNA structure, the optimal choice for the single-strandprotrusion at the 3′ end of the sense strand or antisense strand is TT,UU, AU or UA, and thus varied sequences are obtained. Any one of thesense strands can be used to form a double strand with an antisensestrand, in which the two strands must maintain at least 16, 17, 18, 19,20, 21, 22 or 23 continuous base pairs. Some of the listed sequences maydiffer from the target site by 1, 2 or 3 bases, and the last base at the3′ end of the sense strand may be U, A or T. The last base at 3′ end ofthe antisense strand may be U, A or T. Following the above principle,the following candidate sequences are screened in the present invention:

Targeting position of Sense strand (5′→3′)(withoutAntisense strand (5′→3′) first SEQ ID a protective base moiety such(without a protective base nucleotide No. as TT, UU, UA) SEQ ID No.moiety such as TT, UU, UA) 208 SEQ ID GGGUUUUUCUUGUUGACA SEQ ID NO. 2UUGUCAACAAGAAAAACC NO: 1 A C 209 SEQ ID GGUUUUUCUUGUUGACAA SEQ ID NO. 4UUUGUCAACAAGAAAAAC NO. 3 A C 210 SEQ ID GUUUUUCUUGUUGACAAA SEQ ID NO. 6UUUUGUCAACAAGAAAAA NO. 5 A C 1575 SEQ ID GACCGUGUGCACUUCGCU SEQ ID NO. 8AAGCGAAGUGCACACGGU NOT U C 1576 SEQ ID ACCGUGUGCACUUCGCUU SEQ IDGAAGCGAAGUGCACACGG NO. 9 C NO. 10 U 1577 SEQ ID CCGUGUGCACUUCGCUUCSEQ ID UGAAGCGAAGUGCACACG NO. 11 A NO. 12 G 1578 SEQ IDCGUGUGCACUUCGCUUCA SEQ ID GUGAAGCGAAGUGCACAC NO. 13 C NO. 14 G 1579SEQ ID GUGUGCACUUCGCUUCAC SEQ ID GGUGAAGCGAAGUGCACA NO. 15 C NO. 16 C1580 SEQ ID UGUGCACUUCGCUUCACC SEQ ID AGGUGAAGCGAAGUGCAC NO. 17 U NO. 18A 1677 SEQ ID CAGCAAUGUCAACGACCG SEQ ID GGUCGUUGACAUUGCUGA NO. 19 ANO. 20 A 377 SEQ ID GGAUGUGUCUGCGGCGUU SEQ ID AAACGCCGCAGACACAUC NO. 21U NO. 22 C 376 SEQ ID UGGAUGUGUCUGCGGCGU SEQ ID AACGCCGCAGACACAUCCNO. 23 U NO. 24 A 375 SEQ ID CUGGAUGUGUCUGCGGCG SEQ IDACGCCGCAGACACAUCCA NO. 25 U NO. 26 G 1522 SEQ ID CGGGGCGCACCUCUCUUUSEQ ID UAAAGAGAGGUGCGCCCC NO. 27 A NO. 28 G 1523 SEQ IDGGGGCGCACCUCUCUUUA SEQ ID GUAAAGAGAGGUGCGCCC NO. 29 C NO. 30 C 1525SEQ ID GGCGCACCUCUCUUUACG SEQ ID GCGUAAAGAGAGGUGCGC NO. 31 C NO. 32 C1526 SEQ ID GCGCACCUCUCUUUACGC SEQ ID CGCGUAAAGAGAGGUGCG NO. 33 G NO. 34C 434 SEQ ID UCUUGUUGGUUCUUCUGG SEQ ID UCCAGAAGAACCAACAAG NO. 35 ANO. 36 A 433 SEQ ID UUCUUGUUGGUUCUUCUG SEQ ID CCAGAAGAACCAACAAGA NO. 37G NO. 38 A 435 SEQ ID CUUGUUGGUUCUUCUGGA SEQ ID GUCCAGAAGAACCAACAANO. 39 C NO. 40 G 436 SEQ ID UUGUUGGUUCUUCUGGAC SEQ IDAGUCCAGAAGAACCAACA NO. 41 U NO. 42 A

For the stability of siRNA in tissues, each monomer of the siRNAs ismodified under the conditions of no negative effects or even enhancingits activity. One, two or three incompletely paired bases are allowed inthe sense strand and antisense strand. The nucleotides therein can carrydifferent modifying groups and can be modified in the whole chain or inpart. There may be one or more thio-bases in each strand, even all thebases are thio-bases.

In the compound of the present invention, the modified sense strand andantisense strand are selected from the following sequences:

Targeting position of first SEQ ID No. nucleotide Sense strand (5′→3′)SEQ ID No. Antisense strand (5′→3′) SEQ ID NO. 43 208mGsmGsmGmUmUmUfUmUfCfUfUmG SEQ ID NO. 44 mUsfUsmGmUmCfAmAfCfAfAmGmAmmUmUmGmAmCmAmAsTsT AfAfAfAmCmCmCsmUsmU SEQ ID NO. 45 208mGsmGsmGmUmUmUfUmUfCfUfUmG SEQ ID NO. 46 mUsfUsmGmUfCmAfAfCfAmAmGmAfmUmUmGmAmCmAmAsmUsmU AfAfAmAmCmCmCsmAsmU SEQ ID NO. 47 209mGsmGsmUmUmUmUfUmCfUfUfGmU SEQ ID NO. 48 mUsfUsmUmGmUfCfAfAmCmAmAfGfmUmGmAmCmAmAmAsmAsmU AfAfAmAmAmCmCsmUsmU SEQ ID NO. 49 210mGsmUsmUmUmUmUfCmUfUfGfUmU SEQ ID NO. 50 mUsfUsmUmUmGfUmCfAfAfCmAmAmmGmAmCmAmAmAmAsmUsmU GfAfAfAmAmAmCsmUsmU SEQ ID NO. 51 1575mGsfAsmCfCmGmUmGmUfGfCfAmCm SEQ ID NO. 52 mAsfAsmGmCmGfAmAfGfUfGmCmAmUmUmCmGmCmUmUsmAsmU CfAfCfGmGmUmCsTsT SEQ ID NO. 53 1576mAsmCsmCmGmUfGmUfGfCfAmCmU SEQ ID NO. 54 mGsfAsmAmGmCfGmAfAfGfUmGmCmmUmCmGmCmUmUmCsTsT AfCfAfCmGmGmUsmUsmU SEQ ID NO. 55 1576mAsfCsmCfGmUfGmUfGmCfAmCmUm SEQ ID NO. 56 mGsfAsmAmGmCfGmAfAfGfUmGmCmUmCfGmCmUmUmCsmAsmU AfCfAfCmGmGmUsTsT SEQ ID NO. 57 1577mCsmCsmGmUmGmUfGmCfAfCfUmU SEQ ID NO. 58 mUsfGsmAmAmGfCmGfA fAfGmUmGmCmGmCmUmUmCmAsTsT mCfAfCfAmCmGmGsmUsmU SEQ ID NO. 59 1578mCsmGsmUmGmUmGfCmAfCfUfUmC SEQ ID NO. 60 mGsfUsmGmAmAfGmCfGfAfAmGmUmGmCmUmUmCmAmCsmUsmU mGfCfAfCmAmCmGsmUsmU SEQ ID NO. 61 1579mGsmUsmGmUmGfCmAfCfUfUmCmG SEQ ID NO. 62 mGsfGsmUmGmAfAmGfCfGfAmAmGmCmUmUmCmAmCmCsmAsmU mUfGfCfAmCmAmCsmAsmU SEQ ID NO. 63 1580mUsmGsmUmGmCmAfCmUfUfCfGfCm SEQ ID NO. 64 mAsfGsmGmUmGfAmAfGfCfGmAmAUmUmCmAmCmCmUsmAsmU mGfUfGfCmAmCmAsmAsmU SEQ ID NO. 65 1677mCsmAsmGmCmAmAfUmGfUfCfAmA SEQ ID NO. 66 mGsfGsmUmCmGfUmUfGfAfCmAmUmmCmGmAmCmCmGmAsmAsmU UfGfCfUmGmAmAsmAsmU SEQ ID NO. 67 377mGsmGsmAmUmGmUfGmUfCfUfGmC SEQ ID NO. 68 mAsfAsmAmCmGfCmCfGfCfAmGmAmmGmGmCmGmUmUmUsmAsmU CfAfCfAmUmCmCsTsT SEQ ID NO. 69 376mUsmGsmGmAmUmGfUmGfUfCfUmG SEQ ID NO. 70 mAsfAsmCmGmCfCmGfCfAfGmAmCmmCmGmGmCmGmUmUsmAsmU AfCfAfUmCmCmAsTsT SEQ ID NO. 71 375mCsmUsmGmGmAmUfGmUfGfUfCmU SEQ ID NO. 72 mAsfCsmGmCmCfGmCfAfGfAmCmAmmGmCmGmGmCmGmUsmUsmA CfAfUfCmCmAmGsTsT SEQ ID NO. 73 1522mCsmGsmGmGmGmCfGmCfAfCfCmU SEQ ID NO. 74 mUsfAsmAmAmGfAmGfAfGfGmUmGmCmUmCmUmUmUmAsmUsmA mCfGfCfCmCmCmGsmUsmU SEQ ID NO. 75 1522mCsfGsmGfGmGfCmGfCmAfCmCfUm SEQ ID NO. 76 mUsfAsmAmAmGfAmGfAfGfGmUmGCfUmCfUmUfUmAsmUsmU mCfGfCfCmCmCmGsTsT SEQ ID NO. 77 1523mGsmGsmGmGmCmGfCmAfCfCfUmC SEQ ID NO. 78 mGsfUsmAmAfAmGfAfGmAfGmGmUmUmCmUmUmUmAmCsmUsmA mGfCfGfCmCmCmCsTsT SEQ ID NO. 79 1525mGsmGsmCmGmCmAfCmCfUfCfUmC SEQ ID NO. 80 mGsfCsmGmUmAfAmAfGfAfGmAmGmUmUmUmAmCmGmCsmAsmU mGfUfGfCmGmCmCsTsT SEQ ID NO. 81 1526mGsmCsmGmCmAmCfCmUfCfUfCmU SEQ ID NO. 82 mCsfGsmCmGmUfAmAfAfGfAmGmAmmUmUmAmCmGmCmGsmUsmA GfGfUfGmCmGmCsTsT SEQ ID NO. 83 434mUsmCsmUmUmGmUfUmGfGfUfUmC SEQ ID NO. 84 mUsfCsmCmAmGfAmAfGfAfAmCmCmmUmUmCmUmGmGmAsmAsmU AfAfCfAmAmGmAsmUsmA SEQ ID NO. 85 434mUsmCsfUmUmGmUfUmGfGfUfUmCm SEQ ID NO. 86 mUsfCsmCmAmGfAmAfGfAfAmCmCmUmUmCmUmGmGmAsmUsmA AfAfCfAmAmGmAsmAsmU SEQ ID NO. 87 433mUsmUsmCmUmUfGmUfUfGfGmUmU SEQ ID NO. 88 mCsfCsmAmGmAfAmGfAfAfCmCmAmmCmUmUmCmUmGmGsmAsmU AfCfAfAmGmAmAsmAsmU SEQ ID NO. 89 435mCsmUsmUmGmUmUfGmGfUfUfCmU SEQ ID NO. 90 mGsfUsmCmCmAfGmAfAfGfAmAmCmmUmCmUmGmGmAmCsmAsmU CfAfAfCmAmAmGsmUsmA SEQ ID NO. 91 436mUsmUsmGmUmUfGmGfUfUfCmUmU SEQ ID NO. 92 mAsfGsmUmCmCfAmGfAfAfGmAmAmmCmUmGmGmAmCmUsmAsmU CfCfAfAmCmAmAsmAsmU

wherein:

-   -   mG, mA, mC and mU are 2′-methoxy (2′-OMe) modified nucleotides;        fG, fA, fC and fU are 2′-fluoro modified nucleotides; s is an        inter-nucleoside phosphorothioate bond, the rest of nucleotide        monomers are linked through phosphodiester bonds. In particular:    -   G=guanosine, A=adenosine, U=uridylic acid, C=cytidylic acid, dT        or T=2′-deoxythymidine nucleotide;    -   Gs=3′-thioguanosine, As=3′-thioadenosine, Us=3′-thiouridylic        acid, Cs=3′-thiocytidylic acid, dTs or        Ts=2′-deoxy-3′-thiothymidine nucleotide;    -   mG=2′-O-methylguanosine, mA=2′-O-methyladenosine,        mU=2′-O-methyluridylic acid, mC=2′-O-methylcytidylic acid;    -   mGs=2′-O-methyl-3′-thioguanosine,        mAs=2′-O-methyl-3′-thioadenosine,        mUs=2′-O-methyl-3′-thiouridylic acid,        mCs=2′-O-methyl-3′-thiocytidylic acid;    -   fG=2′-fluoroguanosine, fA=2′-fluoroadenosine,        fU=2′-fluorouridylic acid, fC=2′-fluorocytidylic acid;    -   fGs=2′-fluoro-3′-thioguanosine, fAs=2′-fluoro-3′-thioadenosine,        fUs=2′-fluoro-3′-thiouridylic acid,        fCs=2′-fluoro-3′-thiocytidylic acid.

Further preferably, in some preferable embodiments, in the compound, themodified sense strand and antisense strand are selected from thefollowing sequences:

Targeting SEQ position SEQ ID of first ID NO. nucleotideSense strand 5′→3′ NO. Antisense strand 5′→3′ 43 208mGsmGsmGmUmUmUfUmUfCfUfUmGm 44 mUsfUsmGmUmCfAmAfCfAfAmGmAmAfAfAUmUmGmAmCmAmAsTsT fAmCmCmCsmUsmU 49 210 mGsmUsmUmUmUmUfCmUfUfGfUmUm 50mUsfUsmUmUmGfUmCfAfAfCmAmAmGfAfA GmAmCmAmAmAmAsmUsmU fAmAmAmCsmUsmU 511575 mGsfAsmCfCmGmUmGmUfGfCfAmCmU 52 mAsfAsmGmCmGfAmAfGfUfGmCmAmCfAfCmUmCmGmCmUmUsmAsmU fGmGmUmCsTsT 53 1576 mAsmCsmCmGmUfGmUfGfCfAmCmUmU 54mGsfAsmAmGmCfGmAfAfGfUmGmCmAfCfA mCmGmCmUmUmCsTsT fCmGmGmUsmUsmU 59 1578mCsmGsmUmGmUmGfCmAfCfUfUmCmG 60 mGsfUsmGmAmAfGmCfGfAfAmGmUmGfCfAmCmUmUmCmAmCsmUsmU fCmAmCmGsmUsmU 63 1580 mUsmGsmUmGmCmAfCmUfUfCfGfCmU64 mAsfGsmGmUmGfAmAfGfCfGmAmAmGfUf mUmCmAmCmCmUsmAsmU GfCmAmCmAsmAsmU 751522 mCsfGsmGfGmGfCmGfCmAfCmCfUmCfU 76 mUsfAsmAmAmGfAmGfAfGfGmUmGmCfGfCmCfUmUfUmAsmUsmU fCmCmCmGsTsT 79 1525 mGsmGsmCmGmCmAfCmCfUfCfUmCmU 80mGsfCsmGmUmAfAmAfGfAfGmAmGmGfUf mUmUmAmCmGmCsmAsmU GfCmGmCmCsTsT

The delivery chains consist of a linking chain D, a linker B, a branchedchain L containing a structure for stabilizing steric hindrance and aliver targeting specific ligand X, and the delivery chains arerepresented by formula (II) as below:

When n=1, the formula (II) is:

When n or m=2, the formula (II) is:

When m=3, the formula (II) is:

The liver targeting specific ligand X may be one or morepolysaccharides, polysaccharide derivatives or monosaccharides andmonosaccharide derivatives.

Preferably, the liver targeting specific ligand X is represented byformula (III) as below:

wherein, R₁, R₂ and R₃ are hydrogen or a hydroxy protective group,respectively.

Further preferably, the liver targeting specific ligand X is one or morestructures selected from the group consisting of galactose,galactosamine, N-acetylgalactosamine and the following structures:

wherein, R₁s are one or two groups selected from the group consisting ofOH, NHCOH and NHCOCH₃.

The branched chain L containing a structure for stabilizing sterichindrance is a C3-C18 linear chain containing one or more carbonyl,amido, phosphoryl, oxygen atom or a combination of these groups, and maybe one or more structures selected from the following structures:

wherein, r1 is a positive integer of 1-12, r2 is an integer of 0-20, Zis H or CH₃.

The linker B is selected from the following formulae:

wherein, A₁ is C, O, S or NH; r1 is a positive integer of 1-15, r2 is aninteger of 0-5; A₂ is C, O, S, NH, carbonyl, amido, phosphoryl orthiophosphoryl.

Preferably, the linker B is selected from the following formulae:

wherein, r1, r3, r4 and r5 are a positive integer of 1-15, respectively;r6 is a positive integer of 1-20, r7 is a positive integer of 2-6, r8 isa positive integer of 1-3.

Further preferably, the linker B is selected from the followingstructures:

The linking chain D contains 5 to 20 carbon atoms, and may containamino, carbonyl, amido, oxygen atom, sulphur atom, thiophosphoryl,phosphoryl, cyclic structure or a combination of these groups.

Preferably, the linking chain D is one selected from the followingstructures:

wherein, each n is a positive integer of 1-20, and each n is the same ordifferent positive integer, p is a positive integer of 1-6; s is apositive integer of 2-13; R₁ and R₂ are the same or differentsubstituents represented by a formula selected from the followingstructures: —H, —CH₃, —CH—(CH₃)₂, —CH₂—CH(CH₃)₂, —CH(CH₃)—CH₂—CH₃,—CH₂—C₆H₅, —C₈NH₆, —CH₂—C₆H₄—OH, —CH₂—COOH, —CH₂—CONH₂, —(CH₂)₂—COOH,—(CH₂)₄—NH₂, —(CH₂)₂—CONH₂, —(CH₂)—S—CH₃, —CH₂—OH, —CH(CH₃)—OH, —CH₂—SH,—CH₂—C₃H₃N₂, —(CH₂)₃NHC(NH)NH₂.

Further preferably, the linking chain D is one selected from thefollowing structures:

The delivery chain at the 5′ end of the sense strand of the compoundcarries one or two N-acetylgalactosamines, and is one selected from thefollowing structures:

In some preferable examples, the delivery chain at the 5′ end of thesense strand of the compound is selected from the formulae listed in thetable below:

Code of the delivery chain at the 5′ end of the sense strand Structure5′YICd-01

5′YICc-01

5′ERCd-01

5′ERCc-01

5′YICa-01

5′YICa-02

5′YICa-03

5′YICa-04

5′YICa-05

5′ERCa-01

5′ERCa-02

5′ERCa-03

5′ERCa-04

5 5′ERCa-05

The delivery chain at the 3′ end of the antisense strand of the compoundcarries two or three N-acetylgalactosamines, and the

in the delivery chain is one selected from the following structures:

In some preferable examples, the delivery chain at the 3′ end of theantisense strand of the compound is preferably selected from theformulae listed in the table below:

Code of the delivery chain at the 3′ end of the antisense strandStructure 3′SANCd-01

3′SANCc-01

3′SANCa-01

3′SANCa-02

3′ERCd-01

3′ERCc-01

3′ERCa-01

3′ERCa-02

3′ERCa-03

3′ERCa-04

3′ERCa-05

In some preferable examples, the combination of the delivery chain atthe 5′ end of the sense strand and the delivery chain at the 3′ end ofthe antisense strand of the compound is preferably one of the structuresas shown in the table below:

Code of the combination of delivery Delivery chain at the 5′ end of theNo. chains sense strand  1 GBL-01

 2 GBL-02

 3 GBL-03

 4 GBL-04

 5 GBL-05

 6 GBL-06

 7 GBL-07

 8 GBL-08

 9 GBL-09

10 GBL-10

11 GBL-11

12 GBL-12

13 GBL-13

14 GBL-14

15 GBL-15

16 GBL-16

Delivery chain at the 3′ end of the No. antisense strand  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

In some preferable examples, the compound of the present inventioncomprises the sense strands with delivery chains linked at the 5′ endand the antisense strands with delivery chains linked at the 3′ end, asshown in the table below:

Code of Code of the Sense strand with a delivery chainAntisense strand with a delivery chain siRNA with combination 5′→3′5′→3′ delivery of delivery Code Sequence Code Sequence chains chainsKys-01 5′YICd-01-R ₁- Kyas-01 mUsfUsmUmUmGfUmCfAfAfCm Ky-0101 GBL-01mGsmUsmUmUmUmUfC AmAmGfAfAfAmAmAmCsmUsm mUfUfGfUmUmGmAmC U-R ₂-3′SANCd-01 mAmAmAmAsmUsmU (SEQ ID NO: 50) (SEQ ID NO: 49) Kys-025′YICc-01-R ₁- Kyas-02 mUsfUsmGmUmCfAmAfCfAfAm Ky-0202 GBL-02mGsmGsmGmUmUmUfU GmAmAfAfAfAmCmCmCsmUsm mUfCfUfUmGmUmUmG U-R ₂-3′SANCc-01 mAmCmAmAsTsT (SEQ ID NO: 44) (SEQ ID NO: 43) Kys-035′ERCd-01-R ₁- Kyas-03 mAsfAsmGmCmGfAmAfGfUfGm Ky-0303 GBL-09mGsfAsmCfCmGmUmGm CmAmCfAfCfGmGmUmCsTsT-R ₂ UfGfCfAmCmUmUmCmG -3′ERCd-01mCmUmUsmAsmU (SEQ ID NO: 52) (SEQ ID NO: 51) Kys-04 5′ERCc-01-R ₁-Kyas-04 mGsfAsmAmGmCfGmAfAfGfUm Ky-0404 GBL-10 mAsmCsmCmGmUfGmUfGmCmAfCfAfCmGmGmUsmUsm GfCfAmCmUmUmCmGm U-R ₂ -3′ERCc-01 CmUmUmCsTsT(SEQ ID NO: 54) (SEQ ID NO: 53) Kys-05 5′YICa-01-R ₁- Kyas-05mGsfUsmGmAmAfGmCfGfAfAm Ky-0505 GBL-03 mCsmGsmUmGmUmGfCGmUmGfCfAfCmAmCmGsmUsm mAfCfUfUmCmGmCmUm U-R ₂ -3′SANCa-01 UmCmAmCsmUsmU(SEQ ID NO: 60) (SEQ ID NO: 59) Kys-06 5′ERCa-01-R ₁- Kyas-06mAsfGsmGmUmGfAmAfGfCfGm Ky-0606 GBL-11 mUsmGsmUmGmCmAfCAmAmGfUfGfCmAmCmAsmAsm mUfUfCfGfCmUmUmCm U-R ₂ -3′ERCa-01 AmCmCmUsmAsmU(SEQ ID NO: 64) (SEQ ID NO: 63) Kys-07 5′YICr-01-R ₁- Kyas-07mUsfAsmAmAmGfAmGfAfGfGm Ky-0707 GBL-08 mCsfGsmGfGmGfCmGfCUmGmCfGfCfCmCmCmGsTsT-R ₂ - mAfCmCfUmCfUmCfUm 3′SANCr-01 UfUmAsmUsmU(SEQ ID NO: 76) (SEQ ID NO: 75) Kys-08 5′ERCr-01-R ₁- Kyas-08mGsfCsmGmUmAfAmAfGfAfGm Ky-0808 GBL-16 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′ERCr-01 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-09 5′YICa-02-R ₁- Kyas-09mGsfCsmGmUmAfAmAfGfAfGm Ky-0909 GBL-04 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′SANCa-01 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-10 5′YICa-03-R ₁- Kyas-10mGsfCsmGmUmAfAmAfGfAfGm Ky-1010 GBL-05 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′SANCa-01 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-11 5′YICa-04-R ₁- Kyas-11mGsfCsmGmUmAfAmAfGfAfGm Ky-1111 GBL-06 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′SANCa-02 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-12 5′YICa-05-R ₁- Kyas-12mGsfCsmGmUmAfAmAfGfAfGm Ky-1212 GBL-07 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′SANCa-02 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-13 5′ERCa-02-R ₁- Kyas-13mGsfCsmGmUmAfAmAfGfAfGm Ky-1313 GBL-12 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′ERCa -02 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-14 5′ERCa-03-R ₁- Kyas-14mGsfCsmGmUmAfAmAfGfAfGm Ky-1414 GBL-13 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′ERCa-03 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-15 5′ERCa-04-R ₁- Kyas-15mGsfCsmGmUmAfAmAfGfAfGm Ky-1515 GBL-14 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′ERCa-04 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79) Kys-16 5′ERCa-05-R ₁- Kyas-16mGsfCsmGmUmAfAmAfGfAfGm Ky-1616 GBL-15 mGsmGsmCmGmCmAfCAmGmGfUfGfCmGmCmCsTsT-R ₂ mCfUfCfUmCmUmUmUm -3′ERCa-05 AmCmGmCsmAsmU(SEQ ID NO: 80) (SEQ ID NO: 79)

In some preferable examples, the compound of the present invention has astructure shown in the table below:

Code of siRNA with Code of delivery chains R₁ R₂ compound Ky-0101—NH(CH₂)₅CH₂—

GBL-0401 Ky-0202 —NH(CH₂)₅CH₂—

GBL-0402 Ky-0303 —NH(CH₂)₅CH₂—

GBL-0403 Ky-0404 —NH(CH₂)₅CH₂—

GBL-0404 Ky-0505 —NH(CH₂)₅CH₂—

GBL-0405 Ky-0606 —NH(CH₂)₅CH₂—

GBL-0406 Ky-0707 —NH(CH₂)₅CH₂—

GBL-0407 Ky-0808 —NH(CH₂)₅CH₂—

GBL-0408 Ky-0101 —NH(CH₂)₅CH₂—

GBL-0409 Ky-0101 —NH(CH₂)₅CH₂—

GBL-0410 Ky-0909 —NH(CH₂)₅CH₂—

GBL-0411 Ky-1010 —NH(CH₂)₅CH₂—

GBL-0412 Ky-1111 —NH(CH₂)₅CH₂—

GBL-0413 Ky-1212 —NH(CH₂)₅CH₂—

GBL-0414 Ky-1313 —NH(CH₂)₅CH₂—

GBL-0415 Ky-1414 —NH(CH₂)₅CH₂—

GBL-0416 Ky-1515 —NH(CH₂)₅CH₂—

GBL-0417 Ky-1616 —NH(CH₂)₅CH₂—

GBL-0418.

In another aspect, the present invention provides an application of thecompound of the present invention in the preparation of a medicament fortreating liver-related diseases, wherein, the liver-related diseasesinclude acute and chronic hepatitis, liver cancer, hereditaryliver-derived diseases, liver cirrhosis, fatty liver, diabetes.

In a further aspect, the present invention provides an application ofthe compound of the present invention in the preparation of a medicamentfor treating HBV infection-related diseases, wherein, the HBV infectionincludes chronic hepatitis B virus infection, acute hepatitis B virusinfection.

Among others, the liver targeting specific ligand X is specific againstasialoglycoprotein receptors (ASGPR) in liver, the HBV infection-relateddisease is chronic hepatitis B, and the compound can continuouslyinhibit the expression of HBsAg and HBeAg of HBV and HBV DNA.

In yet another aspect, the present invention provides a pharmaceuticalcomposition, which comprises the compound of the present invention andpharmaceutically acceptable auxiliary materials, and its dosage form ispreferably subcutaneous injection.

Compared to the prior art, the present invention has the followingbeneficial effects:

-   -   (1) Compared to liposome-mediated siRNA delivery: In        liposome-mediated siRNA delivery, liposome mainly encapsulates        siRNA within it to protect siRNA from nuclease degradation, thus        improving the efficiency of siRNA passing the cell membrane        barriers and promoting the uptake of cells. Liposome includes,        e.g., anionic liposomes, pH-sensitive liposomes,        immunoliposomes, fusogenic liposomes and cationic liposomes and        the like. Although some progresses had been made, liposomes        themselves are prone to triggering an inflammatory response, so        before administration of a liposome, various antihistamine and        hormone drugs, such as Cetirizine and dexamethasone, must be        used so as to reduce acute inflammatory responses that may        occur. Therefore, liposomes are not suitable for all therapeutic        areas in practical clinical applications, especially diseases        with long treatment cycles such as chronic hepatitis B, and the        cumulative toxicity that may be generated from long-term use is        a potential safety hazard.    -   (2) A completely new manner for introduction of        N-acetylgalactosamine:

Comparison with siRNAs with three N-acetylgalactosamine moieties interms of effect of HBsAg of HBV suppression: the siRNA drugs currentlyin phase I/II for the treatment of chronic hepatitis B include ARO-HBVand ALN-HBV02. In ARO-HBV, three N-acetylgalactosamine moieties areintroduced through a linking chain at the 5′ end of the sense strand ofthe siRNA, while in ALN-HBV02, three N-acetylgalactosamine moieties areintroduced through a linking chain at the 3′ end of the sense strand ofthe siRNA. In both of the above drugs, the sites for introducinggalactosomines are on the sense strands, and threeN-acetylgalactosamines are introduced. In the compound provided in thepresent invention, different or same numbers of N-acetylgalactosaminesare introduced at the 5′ end of the sense strand and the 3′ end of theantisense strand of siRNA at the same time. So far, no report has beenpublished about introduction at both of the 5′ end of the sense strandand the 3′ end of the antisense strand, especially introduction of threeN-acetylgalactosamines at the 3′ end of the antisense strand, which is acompletely new introduction manner. It has been demonstrated throughexamples that, such an introduction manner allows siRNA to efficientlyinhibit HBV gene.

BRIEF DESCRIPTION OF THE DRAWINGS

To make the objectives, technical solutions and beneficial effects ofthe present invention more clear, a brief description of the attacheddrawings is provided as below:

FIG. 1 is a high-resolution mass spectrum of 5′YICd-01-c4;

FIG. 2 is a high-resolution mass spectrum of 5′YICc-01-c7;

FIG. 3 is a high-resolution mass spectrum of 5′ERCd-01-c7;

FIG. 4 is a high-resolution mass spectrum of 5′ERCc-01-c4;

FIG. 5 is a high-resolution mass spectrum of 3′SANCd-01-c6;

FIG. 6 is a histogram showing in vitro inhibition effect on HBsAg inHepG2.215 cells;

FIG. 7 is a histogram showing in vitro inhibition effect on HBeAg inHepG2.215 cells;

FIG. 8 is a histogram showing in vitro inhibition effect on HBV DNA inHepG2.215 cells;

FIG. 9 is a histogram showing in vivo inhibition effect on HBV gene inTransgenic Mice;

FIG. 10 is a diagram showing in vivo inhibition effect on HBV HBsAg byGBL-0401 in Transgenic Mice.

DETAILED DESCRIPTION

The following examples illustrate some embodiments disclosed in thepresent invention, but the present invention is not limited thereto. Inaddition, when providing specific embodiments, the inventors anticipatedapplication of some specific embodiments, for example, compounds withspecifically same or similar chemical structures for treatment ofdifferent liver-derived diseases.

Explanations

-   -   DMF refers to N,N-dimethylformamide;    -   HBTU refers to O-benzotriazole-tetramethylurea        hexafluorophosphate;    -   DIPEA (DIEA) refers to N,N-diisopropylethylamine;    -   DCM refers to dichloromethane;    -   DMAP refers to 4-dimethylaminopyridine;    -   DMT-CL refers to 4,4′-dimethoxytriphenylchloromethane;    -   THF refers to tetrahydrofuran;    -   TBTU refers to O-benzotriazol-N,N,N′,N′-tetramethylurea        tetrafluoroborate;    -   DBU refers to 1,8-diazabicycloundec-7-ene;    -   HOBt refers to 1-hydroxybenzotrizole;    -   DCC refers to dicyclohexylcarbodiimide;    -   Pd—C refers to palladium-carbon catalyst;    -   refers to a solid phase carrier, such as a resin.

Example 1. Synthesis of GBL-0401 1. Synthesis of Kys-01 1.1. Compoundsof 5′YICd-01: Synthesis of 5′YICd-01-PFP 1.1.1. Synthesis of5′YICd-01-c1

Into 2-hydroxyethylamine (5.0 g, 81.9 mmol), were added 50 mL ofdimethyl sulfoxide and 5 mL of a sodium hydroxide solution at aconcentration of 1 g/mL, followed by dropwise addition of 12 mL oftert-butyl acrylate (81.9 mmol) within 1 hour. The mixture was reactedat room temperature for 24 h, and then 100 mL of petroleum ether wasadded, and the mixture was washed with saturated brine twice. Theorganic layer was dried and passed over a column to get 7.5 g ofcolorless oil.

1.1.2. Synthesis of 5′YICd-01-c2

Into 5′YICd-01-c1 (7.5 g, 39.7 mmol), were added 50 mL of DCM and 23 mLof a sodium carbonate solution (25%), followed by dropwise addition ofbenzyl chloroformate (7.7 g, 45.0 mmol) at room temperature. The mixturewas reacted at room temperature overnight, washed with saturated brinetwice, dried over anhydrous sodium sulfate, and evaporated off thesolvent. The residue was passed over a chromatographic column (ethylacetate:petroleum ether=15%-30%) to get 11.3 g of an oil.

1.1.3 Synthesis of 5′YICd-01-c3

5′YICd-01-c2 (11.3 g, 35.0 mmol) was added with 20 mL of formic acid,and reacted at room temperature overnight. The solvent was evaporatedoff at reduced pressure to get 9.2 g of 5′YICd-01-c3.

1.1.4. Synthesis of 5′YICd-01-c4

1.0 g (3.73 mmol) of 5′YICd-01-c3 and 2.0 g (4.48 mmol) of dlSANC-c4were added into 30 mL of DMF, then added with 0.38 g of HOBt and 2.30 gof HBTU, followed by slow addition of 1.0 mL of DIEA. The mixture wasadded with 20 mL of water and extracted with 40 mL of DCM. The organicphase was washed with 100 mL of saturated brine, dried over anhydroussodium sulfate, and evaporated at reduced pressure to dryness. Theresidue was purified by chromatography on a silica gel column (Eluent:1-15% methanol in DCM) to get 2.2 g of a white foamy solid, of which thehigh-resolution mass spectrum is shown in FIG. 1 .

1.1.5. Synthesis of 5′YICd-01-c5

2.2 g (3.2 mmol) of 5′YICd-01-c4 was dissolved in 30 mL of methanol,added with 1.0 g of 10% Pd—C (wet Degussa-type E101 NE/W), andhydrogenated at normal pressure overnight. The reaction mixture wasfiltered with diatomite, and the filtrate was evaporated at reducedpressure to dryness to get 1.70 g of white foam.

1.1.6. Synthesis of 5′YICd-01-c6

0.80 g (3.60 mmol) of monobenzyl glutarate was weighed and dissolved in2 mL DMF, added with 1.28 g of TBTU and 2.0 mL of DIEA, reacted withstirring for 5 minutes, and then added with 1.70 g (3.0 mmol) of5′YICd-01-c5, and reacted at room temperature with stirring overnight.The reaction solution was evaporated at reduced pressure, added with 50mL of DCM and 50 mL of water and stirred for 5 minutes. The layers wereseparated, and the organic layer was dried over anhydrous sodiumsulfate, passed over a chromatographic column (Eluent:DCM:methanol=1%-10%), and the solvent was evaporated at reduced pressureto get 2.1 g of a white product.

1.1.7. Synthesis of 5′YICd-01-c7

Into a 100 mL single-necked flask, were added 2.1 g (2.7 mmol) of5′YICd-01-c6 and 0.2 g of palladium-carbon. The flask was evacuated by awater pump and supplemented with hydrogen in triplicate. The reactionwas conducted under pressurized hydrogen overnight. On the next day, TLCshowed that the reaction was completed. Palladium-carbon was filteredwith diatomite, and the filtrate was evaporated at reduced pressure toget 1.8 g of a product.

1.1.8. Synthesis of 5′YICd-01-PFP

Into a 100 mL single-necked flask, were added 1.8 g (2.66 mmol) of5′YICd-01-c7 and 20 mL of DCM. 1.1 g (4.0 mmol) of pentafluorophenyltrifluoromethanesulfonate was dropwise added, and reacted at roomtemperature for 1 hour. The reaction mixture was washed with 40 mL ofwater and 10 mL of saturated sodium bisulfite. The organic layer wasdried over anhydrous sodium sulfate and evaporated at reduced pressureto dryness to get 2.3 g of a product.

1.2. Solid-phase synthesis of C6NH-S-01 With mG as the initiationmonomer and with C6NH phosphoramidite monomer as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The solid-phase phosphoramiditemethod includes the following basic steps: 1) deprotection: removing theprotective group (DMT) on the oxygen atom of the solid phase carrier; 2)coupling: adding a first nucleotide monomer, coupling in the directionof 3′ to 5′; 3) oxidation: oxidizing the resulting nucleoside phosphiteinto a more stable nucleoside phosphate (that is, oxidization oftrivalent phosphorus to pentavalent phosphorus); 4) blocking: blocking5′-OH of the nucleotide monomer unreacted in the previous step bycapping to prevent it from reacting further; the above steps wererepeated until the desired sequence was achieved. After beingsynthesized, the ester bond for linking the compound to the initialnucleoside on the solid phase carrier was cleaved with methylamineethanol solution and aqueous ammonia, and protective groups on variousbases and phosphoric acid on the oligonucleotide, including cyanoethyl(P), benzoyl (mA, fA), acetyl (mC, fC), isobutyryl (mG, fG) and4-methoxy triphenylmethyl (C6NH), were removed. The product was purifiedby HPLC, filtered and sterilized, and freeze-dried.

1.3. Liquid-Phase Synthesis of Kys-01 1.3.1. Synthesis of Kys-01-c1

The purified and freeze-dried C6NH-S-01 (12.5 mg) was weighed andcompletely dissolved in a sodium borate buffer (650 μL, 0.06 mol/L).5′YICd-01-PFP (10.3 mg) was weighed and dissolved in dimethyl sulfoxide(100 μL), and added into C6NH-S-01 and mixed uniformly, followed byaddition of N-methylmorpholine (5 μL). The reaction mixture wasultrasonicated at room temperature for 3 h, and purified over a C18column after HPLC detection showed the completion of the reaction.

1.3.2. Synthesis of Kys-01

The purified Kys-01-c1 (32 mL, 5 mg) was taken into 25% hydrazinehydrate (16 mL), mixed uniformly, ultrasonicated at room temperature for10 min, and purified through a C18 column after HPLC detection showedthe completion of the reaction. The product was then freeze-dried to getKys-01 (2 mg) as a white freeze-dried powder.

2. Synthesis of Kyas-01 2.1. Compounds of 3′SANCd-01: Synthesis of3′SANCd-01 Resin 2.1.1. Synthesis of 3′SANCd-01-c1

3-amino-propanediol (9.114 g, 0.100 mol) was weighed and dissolved inTHF (50 mL), cooled, dropwise added with ethyl trifluoroacetate (15.62g, 0.110 mol), and reacted at room temperature for 1 h. The reactionsolution was rotary evaporated to get crude 3′SANCd-01-c1 (18.871 g).

2.1.2. Synthesis of 3′SANCd-01-c2

3′SANCd-01-c1 (5.480 g, 0.030 mol) was dissolved in pyridine (30 mL) andcooled, added with DMT-CL (10.423 g, 0.031 mol) batchwise, reacted indark overnight, and then rotary evaporated to remove pyridine. Theresidue was dissolved in CH₂Cl₂ (50 mL), and washed with saturated brine(50 mL). The organic phase was dried over anhydrous sodium sulfate,filtered and rotary evaporated. The residue was passed over a column toget the product 3′SANCd-01-c2 (10.805 g).

2.1.3. Synthesis of 3′SANCd-01-c3

3′SANCd-01-c2 (10.805 g, 0.022 mol) was dissolved in methanol (60 mL)and THF (30 mL), cooled, dropwise added with a solution of KOH (5.69 g)in water (24 mL), reacted at room temperature for 2 h, and rotaryevaporated to remove methanol and THF. The residue was added with water(50 mL) and extracted with EtOAc (30 mL*3). The organic phase was washedwith saturated brine (50 mL), dried over anhydrous sodium sulfate,filtered, and rotary evaporated. The residue was passed over a columnwith an eluent containing 1% triethylamine to get the product3′SANCd-01-c3 (8.286 g).

2.1.4. Synthesis of 3′SANCd-01-c4

3′SANCd-01-c3(2.890 g, 0.007 mol) was dissolved in CH₂Cl₂ (20 mL) andcooled, dropwise added with a solution of DCC (1.680 g) in CH₂Cl₂ (10mL), stirred for 20 minutes, added with a solution of monomethylsuberate (1.522 g) in CH₂Cl₂ (10 mL), and reacted at room temperatureovernight. The reaction was quenched with 5% NaHCO₃(20 mL) and extractedwith CH₂Cl₂ (20 mL*2). The organic phase was washed with saturated brine(10 mL), dried over anhydrous sodium sulfate, filtered, and rotaryevaporated. The residue was passed over a column with an eluentcontaining 1% triethylamine to get the product 3′SANCd-01-c4 (3.193 g).

2.1.5. Synthesis of 3′SANCd-01-c5

3′SANCd-01-c4 (2.193 g, 0.004 mol) was dissolved in THF (10 mL) andcooled, dropwise added with a solution of LiOH (0.645 g) in water (4.5g) and reacted for 2 h. TLC indicated that there was no raw material.The reaction solution was rotary evaporated to remove the solvent. Theresidue was neutralized with saturated ammonium chloride, and extractedwith CH₂Cl₂ (20 mL*2). The organic phase was washed with saturated brine(10 mL), dried over anhydrous sodium sulfate, filtered, and rotaryevaporated. The residue was passed over a column with an eluentcontaining 1% triethylamine to get the product 3′SANCd-01-c5 (1.979 g).

2.1.6. Synthesis of 3′SANCd-01-c6

3′SANCd-01-c5 (0.389 g, 0.004 mol) was dissolved in DMF (2 mL) andcooled, added with DIPEA (0.15 mL) and TBTU (0.183 g), stirred for 10minutes, added with a solution of dlSANC-c12 (0.756 g, 0.0005 mol) inDMF (2 mL), and reacted at room temperature overnight. The reaction wasquenched with water (20 mL) and extracted with CH₂Cl₂ (20 mL*2). Theorganic phase was washed with saturated brine (10 mL), dried overanhydrous sodium sulfate, filtered, and rotary evaporated. The residuewas passed over a column with an eluent containing 5% triethylamine toget the product 3′SANCd-01-c6 (0.803 g), of which the high-resolutionmass spectrum is shown in FIG. 5 .

2.1.7. Synthesis of 3′SANCd-01-c7

Into a reaction flask, 3′SANCd-01-c6 (2.15 g 0.001 mol) and 22 mL of DCMwere added in order and dissolved with stirring at room temperature, andthen added with DBU (0.156 g) and succinic anhydride (0.3 g, 0.003 mmol)in order, and reacted with stirring at room temperature. TLC analysisshowed the reaction was completed. The reaction mixture was concentratedto remove DCM, and then added with water and extracted with DCM. Theorganic phase was further washed with saturated brine and dried overanhydrous sodium sulfate, filtered, and concentrated. Finally theresidue was purified over a silica gel column to get 2.03 g of3′SANCd-01-c7.

2.1.8. Synthesis of 3′SANCd-01 Resin

Into a reaction flask, 3′SANCd-01-c7 (1.13 g, 0.0005 mmol) and 12 mL ofDMF were added in order and dissolved with stirring at room temperature,added with HBTU (0.11 g), DIPEA (0.104 g) and GE resin (1.80 g) inorder, and shaken in a shaker at 35° C. for 24 h. The mixture wastransferred into a synthesis tube and filtered. Under bubbled withnitrogen, the resin was rinsed with DMF for 4 times. Then CAP A+CAP Bwere added to conduct the end-capping reaction for half an hour underbubbling with nitrogen. A little amount of resin was taken for a kaisertest until the test solution appeared yellow. After completion of theend-capping, the filter cake was rinsed with methanol, DCM and methanol,respectively, and dried in vacuum to get 2.48 g of 3′SANCd-01 resin, ofwhich the degree of substitution was 150 μmol/g.

2.2 Solid-Phase Synthesis of Kyas-01

With mU as the initiation monomer and with MU as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The solid-phase phosphoramiditemethod includes the following basic steps: 1) deprotection: removing theprotective group (DMT) on the oxygen atom of 3′SANCd-01 resin; 2)coupling: adding a first nucleotide monomer, coupling in the directionof 3′ to 5′; 3) oxidation: oxidizing the resulting nucleoside phosphiteinto a more stable nucleoside phosphate (that is, oxidization oftrivalent phosphorus to pentavalent phosphorus); 4) blocking: blocking5′-OH of the nucleotide monomer unreacted in the previous step bycapping to prevent it from reacting further; the above steps wererepeated until the desired sequence was achieved. After beingsynthesized, the ester bond for linking the compound to the initialnucleoside on the solid phase carrier was cleaved with methylamineethanol solution and aqueous ammonia, and protective groups on variousbases and phosphoric acid on the oligonucleotide, including cyanoethyl(P), benzoyl (mA, fA), acetyl (mC, fC) and isobutyryl (mG, fG), wereremoved. The product was purified by HPLC, filtered and sterilized, andfreeze-dried to get Kyas-01.

3. Synthesis of GBL-0401

Kys-01 and Kyas-01 solutions were determined accurately for theirconcentration, mixed at equal molarity, added with 1 M PBS solution at1/20 of the volume and mixed uniformly again. The mixed system washeated to 95° C. for 5 min, cooled naturally for 3 h to 40° C. or roomtemperature, and detected by HPLC. If the single-strand residue was <5%,the reaction is considered complete.

Example 2. Synthesis of GBL-0402 1. Synthesis of Kys-02 1.1. Compoundsof 5′YICc-01: Synthesis of 5′YICc-01-PFP 1.1.1. Synthesis of5′YICc-01-c1

SANC-c8 (7.0 g, 40.0 mmol) and 5′YICd-01-c3 (9.2 g, 34.4 mmol) weredissolved in 25 mL of DMF, added with 9.0 g TBTU and cooled to 10° C.,then added with 2 mL of DIEA and reacted at room temperature overnight.30 mL of water and 50 mL of dichloromethane were added. The organiclayer was washed with saturated brine for three times, dried, andevaporated at reduced pressure to dryness. The residue was passed over achromatographic column (Eluent: dichloromethane:methanol=1%-10%) to get10.0 g of a yellow sticky solid.

1.1.2. Synthesis of 5′YICc-01-c2

15 mL of concentrated hydrochloric acid was added into 10.0 g of5′YICc-01-c1. The mixture was reacted at room temperature overnight, andthen evaporated at reduced pressure to get 7.3 g of a product.

1.1.3. Synthesis of 5′YICc-01-c3

5′YICc-01-c2 (7.3 g, 22.6 mmol) and SANC-c4 (12.1 g, 27.1 mmol) wereadded into 60 mL of DMF, added with 3.8 g of HOBt and 12.4 g of HBTU,followed by slow addition of 5.0 ml of DIEA. The reaction solution wasreacted at room temperature with stirring overnight. Then 50 mL of waterwas added, and the reaction solution was extracted with 100 mL ofdichloromethane. The organic phase was washed with 100 mL of saturatedbrine, dried over anhydrous Na₂SO₄, and evaporated at reduced pressureto dryness. The residue was purified by chromatography on a silica gelcolumn (Eluent:3-15% MeOH in DCM) to get 8.3 g of a white foamy solid.

1.1.4. Synthesis of 5′YICc-01-c7

The synthetic steps were the same as those in 1.1.5 of Example 1, andthe high-resolution mass spectrum is shown in FIG. 2 .

1.1.5. Synthesis of 5′YICc-01-c8

The synthetic steps were the same as those in 1.1.6 of Example 1.

1.1.6. Synthesis of 5′YICc-01-c9

The synthetic steps were the same as those in 1.1.7 of Example 1.

1.1.7. Synthesis of 5′YICc-01-PFP

The synthetic steps were the same as those in 1.1.8 of Example 1.

1.2. Solid-Phase Synthesis of C6NH-S-02

With mG as the initiation monomer and with C₆NH phosphoramidite monomeras the end monomer, different phosphoramidite monomers were introducedby coupling through a solid-phase phosphoramidite method. The syntheticsteps were the same as those in 1.2 solid-phase synthesis of Example 1.

1.3. Liquid-Phase Synthesis of Kys-02 1.3.1. Synthesis of Kys-02-c1

The synthetic steps were the same as those in 1.3.1 of Example 1.

1.3.2. Synthesis of Kys-02

The synthetic steps were the same as those in 1.3.2 of Example 1.

2. Synthesis of Kyas-02 2.1. Compounds of 3′SANCc-01: Synthesis of3′SANCc-01 Resin

The synthetic route and process steps of 3′SANCc-01 resin wereconsistent with those of 3′SANCd-01 resin, except the synthesis of3′SANCc-01-c6.

2.1.1. Synthesis of 3′SANCc-01-c1

3′SANCd-01-c5 (0.295 g) was dissolved in DMF (2 mL) and cooled, addedwith DIPEA (0.14 mL) and TBTU (0.177 g) and stirred for 10 minutes, thenadded with a solution of SANC-c12 (0.756 g) in DMF (2 mL), and reactedat room temperature overnight. The system was quenched with water (50mL) and extracted with CH₂Cl₂ (20 mL*2). The organic phase was washedwith saturated brine (10 mL), dried over anhydrous sodium sulfate,filtered, and rotary evaporated. The residue was passed over a columnwith an eluent containing 5% triethylamine to get the product3′SANCc-01-c6 (0.815 g).

2.2. Solid-Phase Synthesis of Kyas-02

With mU as the initiation monomer and with mU as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The synthetic steps were the same asthose in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.

3. Synthesis of GBL-0402

Kys-02 and Kyas-02 solutions were determined accurately for theirconcentration. The synthetic steps were the same as those in 3.Synthesis of GBL-0401 in Example 1.

Example 3. Synthesis of GBL-0403 1. Synthesis of Kys-03 1.1. Compoundsof 5′ERCd-01: Synthesis of 5′ERCd-01-PFP 1.1.1. Synthesis of5′ERCd-01-c1

5.0 g (54.9 mmol) of 2-amino-1,3-propanediol was weighed, added with 50mL of DMSO and 5 mL of a solution of sodium hydroxide at a concentrationof 1 g/mL and cooled to 0° C., dropwise added with 20 mL (137.8 mol) oftert-butyl acrylate over 2 hours and reacted at room temperature for 48h. The mixture was added with 100 mL petroleum ether. The organic phasewas washed with saturated brine twice, dried and passed over achromatographic column (Eluent: ethyl acetate:petroleum ether=25%-75%containing 0.05% triethylamine) to get 6.2 g of a colorless oil.

1.1.2. Synthesis of 5′ERCd-01-c2

5′ERCd-01-c1 (6.2 g, 17.9 mmol) was weighed, added with 50 mL ofdichloromethane and 23 mL of a sodium carbonate solution (25%), followedby dropwise addition of 8.2 mL (57.4 mmol) of benzyl chloroformate atroom temperature over 2 hours. The mixture was reacted at roomtemperature overnight, washed with saturated brine for three times,dried over anhydrous sodium sulfate, and evaporated off the solvent. Theresidue was passed over a chromatographic column (ethylacetate:petroleum ether=5%-30%) to get 4.0 g of an oil.

1.1.3. Synthesis of 5′ERCd-01-c3

4.0 g (8.3 mmol) of 5′ERCd-01-c2 was added with 12 mL of formic acid,reacted at room temperature overnight, and evaporated off the solvent atreduced pressure to get 2.8 g of 5′ERCd-01-c3.

1.1.4. Synthesis of 5′ERCd-01-c4

5′ERCd-01-c3 (1.11 g, 3.0 mmol) and dlSANC-c4 (3.6 g, 8.04 mmol) wereadded into 60 mL of DMF, added with 2.24 g of HOBt and 3.36 g of HBTU,followed by slow addition of 4.16 mL of DIEA. The reaction solution wasreacted with stirring at room temperature for 3 hours. Water was thenadded, and the aqueous layer was extracted with dichloromethane (2×10mL). The organic layer was combined, and then washed with 80 mL ofsaturated NaHCO₃, water (2×60 mL), and saturated brine (60 mL) in order,dried over anhydrous Na₂SO₄, and evaporated at reduced pressure todryness. The residue was purified by chromatography on a silica gelcolumn (Eluent: 3-15% MeOH in DCM), to get 3.24 g of a light yellowsolid.

1.1.5. Synthesis of 5′ERCd-01-c5

3.24 g (2.6 mmol) of 5′ERCd-01-c4 was dissolved in 60 mL of methanol,added with 0.3 g of 10% Pd—C(wet Degussa-type E101 NE/W) and 2.0 mL ofacetic acid, and hydrogenated at normal pressure overnight. The reactionsolution was filtered with diatomite, and the filtrate was evaporated atreduced pressure to get 2.9 g of an oil.

1.1.6. Synthesis of 5′ERCd-01-c6

0.21 g (0.001 mol) of monobenzyl glutarate was weighed and dissolved in2 mL of DM1F, added with 0.36 g of TBTU and 0.4 mL of DIEA, reacted withstirring for 5 minutes, added with 0.50 g of 5′ERCd-01-c5 (dissolved in10 ml DMF), and reacted at room temperature with stirring overnight. Thereaction solution was evaporated at reduced pressure to dryness, and 40mL of dichloromethane and 20 mL of water were added and stirred for 5minutes. The layers were separated. The organic layer was dried overanhydrous sodium sulfate, and passed over a chromatographic column(Eluent: dichloromethane:methanol=1%-10%). The eluate was evaporated offthe solvent at reduced pressure to get 0.51 g of a white product.

1.1.7. Synthesis of 5′ERCd-01-c7

Into a 100 mL single-necked flask, were added 0.51 g (0.42 mmol) of5′ERCd-01-c6 and 127 mg of palladium-carbon. The flask was evacuatedwith a water pump and supplemented with hydrogen in triplicate. Themixture was reacted under pressurized hydrogen overnight. On the nextday, TLC showed that the reaction was complete. Palladium-carbon wasfiltered with diatomite, and the filtrate was evaporated at reducedpressure to dryness to get 0.40 g of a product, of which thehigh-resolution mass spectrum is shown in FIG. 3 .

1.1.8. Synthesis of 5′ERCd-01-PFP

Into a 50 mL single-necked flask, were added 0.40 g (0.33 mmol) of5′ERCd-01-c7 and 10 mL of dichloromethane, and then dropwise added with0.19 g (0.6 mmol) of pentafluorophenyl trifluoromethanesulfonate over 10minutes and reacted at room temperature for 2 hours. The reactionsolution was washed with 10 mL of water twice, and then with 5 mL ofsaturated sodium bisulfate once. The organic layer was dried overanhydrous sodium sulfate for 10 minutes and evaporated at reducedpressure to dryness to get 0.5 g of a product.

1.2. Solid-Phase Synthesis of C6NH-S-03

With mG as the initiation monomer and with C₆NH phosphoramidite monomeras the end monomer, different phosphoramidite monomers were introducedby coupling through a solid-phase phosphoramidite method. The syntheticsteps were the same as those in 1.2 Solid-phase synthesis in Example 1.

1.3. Liquid-Phase Synthesis of Kys-03 1.3.1. Synthesis of Kys-03-c1

The synthetic steps were the same as those in 1.3.1 of Example 1.

1.3.2. Synthesis of Kys-03

The synthetic steps were the same as those in 1.3.2 of Example 1.

2. Synthesis of Kyas-03 2.1. Compounds of 3′ERCd-01: Synthesis of3′ERCd-01 Resin 2.1.1. Synthesis of 3′ERCd-01-c1

Into a reaction flask, 3′SANCd-01-c5 (0.824 g, 0.0015 mol) and 10 mL ofDMF were added in order and dissolved with stirring at room temperature,and then added with TBTU (0.563 g) and DIPEA (0.517 g) in order anddissolved with stirring at room temperature, and finally added withdlERC-c12 (1.09 g, 0.001 mol) and reacted with stirring at roomtemperature overnight. TLC analysis showed the reaction was complete,the reaction mixture was concentrated to remove DMF, added with waterand extracted with DCM. The organic phase was further washed with asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, filtered, and concentrated. Finally the residue waspurified over a silica gel column to get 1.3 g of an off-white foamysolid.

2.1.2. Synthesis of 3′ERCd-01-c2

The synthetic steps were the same as those in 2.1.7 of Example 1.

2.1.3. Synthesis of 3′ERCd-01 Resin

The synthetic steps were the same as those in 2.1.8 of Example 1.

2.2. Solid-Phase Synthesis of Kyas-03

With mA as the initiation monomer and with T as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The synthetic steps were the same asthose in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.

3. Synthesis of GBL-0403

Kys-03 and Kyas-03 solutions were determined accurately for theirconcentration. The synthetic steps were the same as those in 3.Synthesis of GBL-0401 in Example 1.

Example 4. Synthesis of GBL-0404 1. Synthesis of Kys-04 1.1. Compoundsof 5′ERCc-01: Synthesis of 5′ERCc-01-PFP 1.1.1. Synthesis of5′ERCc-01-c1

N-tert-butoxycarbonyl-1,3-propanediamine (5.0 g, 28.7 mmol) and5′ERCd-01-c3 (2.8 g, 7.6 mmol) were dissolved in 25 mL of DMF, addedwith 9.0 g of TBTU and 2 mL of DIEA and reacted at room temperatureovernight. 30 mL of water and 50 mL of DCM were added. The organic layerwas washed with saturated brine and evaporated at reduced pressure todryness. The residue was passed over a chromatographic column loadedwith petroleum ether and rinsed with 1 L petroleum ether (Eluent:DCM:methanol=5%-10%) to get 2.9 g of a yellow sticky solid.

1.1.2. Synthesis of 5′ERCc-01-c2

2.9 g of 5′ERCc-01-c1 was weighed, added with 9 mL of concentratedhydrochloric acid and reacted at room temperature overnight. The mixturewas evaporated at reduced pressure to get 2.7 g of a product.

1.1.3. Synthesis of 5′ERCc-01-c3

5′ERCc-01-c2 (1.56 g, 2.44 mmol) and Sanc-c4 (3.6 g, 8.04 mmol) wereadded into 60 mL of DMF, added with 2.24 g of HOBt and 3.36 g of HBTU,followed by slow addition of 4.16 mL of DIEA. The reaction solution wasreacted at room temperature with stirring for 1 hour. Water was thenadded, and the aqueous layer was extracted with DCM (2×10 mL). Theorganic layer was combined, and then washed with 80 mL of saturatedsodium bicarbonate, 40 mL of water, and 60 mL of saturated brine inorder, dried over anhydrous sodium sulfate, and evaporated at reducedpressure to dryness. The residue was purified by chromatography on asilica gel column (Eluent: 3-15% methanol in DCM), to get 2.36 g of alight yellow solid.

1.1.4. Synthesis of 5′ERCc-01-c4

2.36 g (1.2 mmol) of 5′ERCc-01-c3 was dissolved in 120 mL of methanol,added with 1.0 g of 10% Pd—C(wet Degussa-type E101 NE/W), andhydrogenated at normal pressure overnight. The reaction solution wasfiltered with diatomite, and the filtrate was evaporated at reducedpressure to dryness to get 1.8 g of oil, of which the high-resolutionmass spectrum is shown in FIG. 4 .

1.1.5. Synthesis of 5′ERCc-01-c5

0.21 g (0.001 mol) of monobenzyl glutarate was dissolved in 2 mL of DMF,added with 0.36 g of TBTU and 0.4 mL of DIEA and reacted with stirringfor 5 minutes, and added with 1.09 g of 5′ERCc-01-c4 and reacted at roomtemperature with stirring overnight. The reaction solution wasevaporated at reduced pressure to dryness, added with 40 mL of DCM and20 mL of water and stirred for 5 minutes. The layers were separated, andthe organic layer was dried over anhydrous sodium sulfate and passedover a chromatographic column (Eluent: DCM:methanol=1%-10%), and thesolvent was evaporated at reduced pressure to dryness to get 0.85 g of awhite product.

1.1.6. Synthesis of 5′ERCc-01-c6

Into a 100 mL single-necked flask, were added 0.85 g (0.43 mmol) of5′ERCc-01-c5 and 127 mg of palladium-carbon. The flask was evacuated bya water pump and supplemented with hydrogen in triplicate. The reactionwas conducted under pressurized hydrogen overnight. On the next day, TLCshowed the reaction was complete. Palladium-carbon was filtered withdiatomite, and the filtrate was evaporated at reduced pressure todryness to get 0.76 g of a product.

1.1.7. Synthesis of 5′ERCc-01-PFP

Into a 50 mL single-necked flask, were added 0.76 g (0.40 mmol) of5′ERCc-01-c6 and 10 mL of DCM, and dropwise added with 0.19 g (0.6 mmol)of pentafluorophenyl trifluoromethanesulfonate, reacted at roomtemperature for 1 hour, and washed with 10 mL of water and 5 mL ofsaturated sodium bisulfite in order. The organic layer was dried overanhydrous sodium sulfate for 10 minutes and evaporated at reducedpressure to dryness to get 0.8 g of a product.

1.2. Solid-Phase Synthesis of C6NH-S-04

With mA as the initiation monomer and with C₆NH phosphoramidite monomeras the end monomer, different phosphoramidite monomers were introducedby coupling through a solid-phase phosphoramidite method. The syntheticsteps were the same as those in 1.2 Solid-phase synthesis in Example 1.

1.3. Liquid-Phase Synthesis of Kys-04 1.3.1. Synthesis of Kys-04-c1

The synthetic steps were the same as those in 1.3.1 of Example 1.

1.3.2. Synthesis of Kys-04

The synthetic steps were the same as those in 1.3.2 of Example 1.

2. Synthesis of Kyas-04 2.1. Compounds of 3′ERCc-01: Synthesis of3′ERCc-01 Resin 2.1.1. Synthesis of 3′ERCc-01-c1

Into a reaction flask were added 3′SANCd-01-c5 (0.824 g, 0.0015 mol) and10 mL of DMF in order and dissolved with stirring at room temperature,and then added with TBTU (0.563 g) and DIPEA (0.517 g) in order anddissolved with stirring at room temperature, and finally added withERC-c12 (1.21 g, 0.001 mol) and reacted with stirring at roomtemperature overnight. TLC analysis showed that the reaction wascomplete, the reaction mixture was concentrated to remove DMF, addedwith water and extracted with DCM. The organic phase was further washedwith a saturated aqueous solution of sodium chloride, dried overanhydrous sodium sulfate, filtered and concentrated. Finally the residuewas purified over a silica gel column to get 1.4 g of a white foamysolid.

2.1.2. Synthesis of 3′ERCc-01-c2

The synthetic steps were the same as those in 2.1.7 of Example 1.

2.1.3. Synthesis of 3′ERCc-01 Resin

The synthetic steps were the same as those in 2.1.8 of Example 1.

2.2. Solid-Phase Synthesis of Kyas-04

With mG as the initiation monomer and with mU as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The synthetic steps were the same asthose in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.

3. Synthesis of GBL-0404

Kys-04 and Kyas-04 solutions were determined accurately for theirconcentration. The synthetic steps were the same as those in 3.Synthesis of GBL-0401 in Example 1.

Example 5. Synthesis of GBL-0409 1. Synthesis of Kyas-09 1.1. Compoundsof 3′qfSANCd-01: Synthesis of 3′qfSANCd-01 Resin 1.1.1. Synthesis of3′qfSANCd-01-c1

Into a reaction flask were added hydroxyprolinol hydrochloride (1.53 g,0.01 mol) and 15 mL of DMF in order and dissolved with stirring at roomtemperature, and then added with monomethyl suberate (1.98 g, 0.0105mol), HBTU (4.55 g) and DIPEA (3.88 g) in order, and reacted withstirring at room temperature overnight. TLC analysis showed that thereaction was complete, and the reaction mixture was concentrated toremove DMF, added with water and extracted with DCM. The organic phasewas further washed with a saturated aqueous solution of sodium chloride,dried over anhydrous sodium sulfate, filtered, and concentrated. Finallythe residue was purified over a silica gel column to get 2.38 g of ayellow sticky liquid.

1.1.2. Synthesis of 3′qfSANCd-01-c2

Into a reaction flask were added 3′qfSANCd-01-c1 (2.87 g 0.01 mol) and30 ml of pyridine in order and dissolved with stirring at roomtemperature, and then added with DMAP (0.61 g) and DMT-CL (4.06 g, 0.012mol) in order, and reacted with stirring at room temperature overnight.TLC analysis showed that the reaction was complete, and the reactionmixture was concentrated to remove pyridine, added with water andextracted with DCM. The organic phase was further washed with asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, filtered, and concentrated. Finally the residue waspurified over a silica gel column to get 4.13 g of a yellow stickyliquid (yield 70%).

1.1.3. Synthesis of 3′qfSANCd-01-c3

Into a reaction flask were added 3′qfSANCd-01-c2 (5.89 g 0.01 mol) and60 mL of a solvent (THF/water/methanol=1:1:4) in order and dissolvedwith stirring at room temperature, and then added with LiOH (1.26 g) andreacted with stirring at room temperature for 2 h. TLC analysis showedthat the reaction was complete, and the reaction mixture wasconcentrated to remove the solvent, added with water and extracted withDCM. The organic phase was further washed with a saturated aqueoussolution of sodium chloride, dried over anhydrous sodium sulfate,filtered, and concentrated. Finally the residue was purified over asilica gel column to get 4.5 g of a yellow sticky liquid.

1.1.4. Synthesis of 3′qfSANCd-01-c4

Into a reaction flask were added 3′qfSANCd-01-c3 (0.863 g, 1.5 mmol) and10 mL of DMF in order and dissolved with stirring at room temperature,and then added with TBTU (0.963 g) and DIPEA (0.517 g) in order anddissolved with stirring at room temperature, and finally added withdlSANC-c12 (1.62 g 1 mmol) and reacted with stirring at room temperatureovernight. TLC analysis showed that the reaction was complete, and thereaction mixture was concentrated to remove DMF, added with water andextracted with DCM. The organic phase was further washed with asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, filtered, and concentrated. Finally the residue waspurified over a silica gel column to get 1.743 g of a yellow stickyliquid.

1.1.5. Synthesis of 3′qfSANCd-01-c5

Into a reaction flask were added 3′qfSANCd-01-c4 (2.18 g, 0.001 mol) and10 mL of DCM in order and dissolved with stirring at room temperature,and then added with DBU (0.256 g) and succinic anhydride (0.3 g, 0.003mmol) in order and reacted with stirring at room temperature. TLCanalysis showed that the reaction was complete, and the reaction mixturewas concentrated to remove DCM, added with water and extracted with DCM.The organic phase was further washed with a saturated aqueous solutionof sodium chloride, dried over anhydrous sodium sulfate, filtered, andconcentrated. Finally the residue was purified over a silica gel columnto get 2.05 g of 3′qfSANCd-01-c5.

1.1.6. Synthesis of 3′qfSANCd-01-c6

Into a reaction flask were added 3′qfSANCd-01-c5 (1.14 g, 0.0005 mmol)and 12 mL of DMF in order and dissolved with stirring at roomtemperature, and then added with HBTU (0.19 g), DIPEA (0.194 g) and GEresin (1.83 g) in order, and shaken in a shaker at 35° C. for 4 h. Themixture was transferred into a synthesis tube and filtered. Underbubbling with nitrogen, the resin was rinsed with DMF for 4 times, addedwith CAP A+CAP B to conduct the end-capping reaction for half an hourunder bubbling with nitrogen. A little amount of the resin was taken fora kaiser test until the test solution appeared yellow. After completionof the end-capping, the filter cake was rinsed with methanol and DCMrespectively, and dried in vacuum to get 2.5 g of 3′qfSANCd-01, of whichthe degree of substitution was 140 μmol/g.

1.2 Solid-Phase Synthesis of Kyas-09

With mU as the initiation monomer and with mU as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The synthetic steps were the same asthose in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.

2. Synthesis of GBL-0409

Kys-01 and Kyas-09 solutions were determined accurately for theirconcentration. The synthetic steps were the same as those in 3.Synthesis of GBL-0401 in Example 1.

Example 6. Synthesis of GBL-0410 1. Synthesis of Kys-10 1.1. Solid-PhaseSynthesis of C9NH-S-01

With mU as the initiation monomer and with C9NH phosphoramidite monomeras the end monomer, different phosphoramidite monomers were introducedby coupling through a solid-phase phosphoramidite method. The syntheticsteps were the same as those in 1.2 Solid-phase synthesis of C6NH-S-01in Example 1.

1.2. Liquid-Phase Synthesis of Kys-10 1.2.1. Synthesis of Kys-10-c1

The synthetic steps were the same as those in 1.3.3 of Example 1.

1.2.2. Synthesis of Kys-01

The synthetic steps were the same as those in 1.3.4 of Example 1.

2. Synthesis of Kyas-10 2.1. Compounds of 3′pdSANCd-01: Synthesis of3′pdSANCd-01 Resin 2.1.1. Synthesis of 3′pdSANCd-01-c1

Into a reaction flask, 4,4-piperidinediyl dimethanol (1.59 g, 0.01 mol)and 20 mL of DMF were added in order and dissolved with stirring at roomtemperature, and then added with monomethyl suberate (1.98 g, 0.0105mol), HBTU (4.55 g) and DIPEA (3.88 g) in order and reacted withstirring at room temperature overnight. TLC analysis showed that thereaction was complete, and the reaction mixture was concentrated toremove DMF, added with water and extracted with DCM. The organic phasewas further washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous sodium sulfate, filtered, and concentrated.Finally the residue was purified over a silica gel column to get 2.65 gof a yellow sticky liquid.

2.1.2. Synthesis of 3′pdSANCd-01-c2

Into a reaction flask, 3′pdSANCd-01-c1 (3.29 g, 0.01 mol) and 33 mLpyridine were added in order and dissolved with stirring at roomtemperature, and then added with DMAP (0.61 g) and DMT-CL (4.06 g, 0.012mol) in order and reacted with stirring at room temperature overnight.TLC analysis showed that the reaction was complete, and the reactionmixture was concentrated to remove pyridine, added with water andextracted with DCM. The organic phase was further washed with asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, filtered, and concentrated. Finally the residue waspurified over a silica gel column to get 4.74 g of a yellow stickyliquid.

2.1.3. Synthesis of 3′pdSANCd-01-c3

Into a reaction flask, 3′pdSANCd-01-c2 (3.16 g, 5 mmol) and 32 mL of asolvent (THF/water/methanol=1:1:4) were added in order and dissolvedwith stirring at room temperature, and then added with LiOH (0.63 g) andreacted with stirring at room temperature for 2 h. TLC analysis showedthat the reaction was complete, and the reaction mixture wasconcentrated to remove the solvent, added with water and extracted withDCM. The organic phase was further washed with a saturated aqueoussolution of sodium chloride and dried over anhydrous sodium sulfate,filtered, and concentrated. Finally the residue was purified over asilica gel column to get 2.78 g of a yellow sticky liquid.

2.1.4. Synthesis of 3′pdSANCd-01-c4

Into a reaction flask, 3′pdSANCd-01-c3 (0.93 g, 1.5 mmol) and 10 mL ifDMF were added in order and dissolved with stirring at room temperature,and then added with TBTU (0.963 g) and DIPEA (0.517 g) in order anddissolved with stirring at room temperature, and finally added withdlSANC-c12 (0.562 g, 1 mmol) and reacted with stirring at roomtemperature overnight. TLC analysis showed that the reaction wascomplete, and the reaction mixture was concentrated to remove DMF, addedwith water and extracted with DCM. The organic phase was further washedwith a saturated aqueous solution of sodium chloride, dried overanhydrous sodium sulfate, filtered, and concentrated. Finally theresidue was purified over a silica gel column to get 1.688 g of a yellowsticky liquid.

2.1.5. Synthesis of 3′pdSANCd-01-c5

Into a reaction flask, 3′pdSANCd-01-c4 (2.22 g, 0.001 mol) and 22 mL ofDCM were added in order and dissolved with stirring at room temperature,and then added with DBU (0.256 g) and succinic anhydride (0.3 g, 0.003mmol) in order and reacted with stirring at room temperature. TLCanalysis showed that the reaction was complete, and the reaction mixturewas concentrated to remove DCM, added with water and extracted with DCM.The organic phase was further washed with a saturated aqueous solutionof sodium chloride, dried over anhydrous sodium sulfate, filtered, andconcentrated. Finally the residue was purified over a silica gel columnto get 2.11 g of 3′pdSANCd-01-c5.

2.1.6. Synthesis of 3′pdSANCd-01

Into a reaction flask, 3′pdSANCd-01-c5 (1.16 g, 0.0005 mmol) and 12 mLof DMF were added in order and dissolved with stirring at roomtemperature, and then added with HBTU (0.19 g), DIPEA (0.194 g) and GEresin (1.85 g) in order, and shaken in a shaker at 35° C. for 4 h. Themixture was transferred into a synthesis tube and filtered. Underbubbling with nitrogen, the resin was rinsed with DMF for 4 times, andthen added with CAP A+CAP B to conduct the end-capping reaction for halfan hour under bubbling with nitrogen. A little amount of the resin wastaken for a kaiser test until the test solution appeared yellow Aftercompletion of the end-capping, the filter cake was rinsed with methanoland DCM respectively, and dried in vacuum to get 2.6 g of 3′pdSANCd-01,of which the degree of substitution was 145 μmol/g.

2. Solid-Phase Synthesis of Kyas-10

With mU as the initiation monomer and with mU as the end monomer,different phosphoramidite monomers were introduced by coupling through asolid-phase phosphoramidite method. The synthetic steps were the same asthose in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.

3. Synthesis of GBL-0410

Kys-10 and Kyas-10 solutions were determined accurately for theirconcentration. The synthetic steps were the same as those in of Example1 3, Synthesis of GBL-0401.

Example 7. GBL0405 to GBL0408 and GBL0411 to GBL0418 were SynthesizedReferring to GBL-0401 to GBL0404 Example 8. In Vitro Assay of theInhibition Effects of the Compounds Against HBV Genes in HepG2.2.15Cells 1. Experimental Grouping

Blank control group: Adding a DMEM medium containing 2% FBS andincubating for 72 h.

Test sample groups: A test sample dilution at a concentration of 5 nM,0.5 nM or 0.05 nM was added respectively. Each concentration was done intriplicate. The incubation was conducted in an incubator at 37° C. and5% CO₂ for 72 h.

2. Experimental Materials

HepG2.2.15 cells

3. Experimental Reagents

Name Brand Lot No. DMEM medium with high glucose Gibco 8119164 Fetalbovine serum Gibco 20190907 PBS Solarbio 20190624 Trypsin-EDTA solutionGibco 2062475 Dual antibiotic solution Gibco 2029632(Penicillin/Streptomycin solution) HBsAg, HBeAg kit Shanghai Kehua201812381

4. Experimental Instruments

Name Brand Model No. Biosafety cabinet Haier HR40-IIA2 CO₂ IncubatorASTEC SCA-165DS Ordinary optical microscope Nikon TS2-S-SM Low-speedcentrifuge Flying pigeon KA-1000 Multi-door refrigerator MeiLingBCD-318WTPZM (E)

5. Test Samples

No. Code of new compounds Weight Purity 1 GBL-0401 13.8 μg 92.3% 2GBL-0402 12.9 μg 86.4% 3 GBL-0403 13.4 μg 89.3% 4 GBL-0404 14.0 μg 93.3%5 GBL-0405 13.7 μg 91.3% 6 GBL-0406 20.5 μg 88.3% 7 GBL-0407 20.1 μg94.4% 8 GBL-0408 20.3 μg 92.3% 9 GBL-0409 20.4 μg 93.6% 10 GBL-0410 20.2μg 90.5% 11 GBL-0411 20.0 μg 89.5% 12 GBL-0412 15.1 μg 94.8% 13 GBL-041315.2 μg 92.5% 14 GBL-0414 15.5 μg 90.6% 15 GBL-0415 15.7 μg 91.5% 16GBL-0416 16.0 μg 93.4% 17 GBL-0417 15.9 μg 91.7% 18 GBL-0418 15.5 μg92.5%

6. Test Process

HepG2.2.15 cells were incubated in a 96-well cell culture plate, andfresh medium was replaced every three days. Drug-containing culturemedia with different concentrations formulated above were added on Day6, and the incubation continued until Day 9. The supernatants werecollected and the contents of HBsAg, HbeAg and HBV DNA in the cellsupernatant were detected with a detection kit. The results of OD valueswere compared with that of the control group without administration, andthe effectiveness can be determined according to the ratio.

7. Experimental Results 7.1 Inhibition Effects on HbsAg in HepG2.2.15Cells: See FIG. 6 7.2 Inhibition Effects on HbeAg in the Supernatant ofHepG2.2.15 Cells: See FIG. 7 7.3 Inhibition Effects on HBV DNA in theSupernatant of HepG2.2.15 Cells: See FIG. 8 Example 9. In Vivo Assay onInhibitory Effects of the New Compounds Against HBV Genes in TransgenicMice

1. Experimental Protocol

The experimental assay was performed on male HBV transgenic mice ofproper age (requiring that HBsAg was significantly expressed). 90 miceweighing about 25 g were chosen and randomly divided into 18 groups,with 5 mice in each group. On Day 0, each mouse was administered bysubcutaneous injection at 3 mg/kg with an administration volume of100-200 μL. Before administration, HBsAg in the blood of mice wasdetermined, and the average level of HBsAg in various groups was triedto be kept consistent.

2. Test Samples and Reagents

No. Code of new compounds Specification Purity/Content 1 GBL-0401 500 μg92.3% 2 GBL-0402 500 μg 86.4% 3 GBL-0403 500 μg 89.3% 4 GBL-0405 500 μg93.3% 5 GBL-0406 500 μg 91.3% 6 GBL-0407 500 μg 88.3% 7 GBL-0408 500 μg94.4% 8 GBL-0409 500 μg 92.3% 9 GBL-0410 500 μg 93.6% 10 GBL-0411 500 μg90.5% 11 GBL-0412 500 μg 89.5% 12 GBL-0413 500 μg 94.8% 13 GBL-0414 500μg 92.5% 14 GBL-0414 500 μg 90.6% 15 GBL-0415 500 μg 91.5% 16 GBL-0416500 μg 93.4% 17 GBL-0417 500 μg 91.7% 18 GBL-0418 500 μg 92.5% 19 Normalsaline 500 ml/flask 0.9%

4. Experimental Instruments

Kit Name Lot No. Manufacturer Kit for hepatitis B virus surface 39531900Roche Diagnostics antigen (Electrochemiluminescence) (Shanghai) Ltd. Co.

5. Experimental Results

Name Model No. Manufacturer Vortex blender MIX-28 DragonLAB CentrifugeS1010E THERMO Full-automatic 602 Roche Diagnostics GmbH chemiluminescentanalyzer

The inhibition effects were shown in FIG. 9 .

Example 10. In Vivo Assay on Inhibitory Effect of GBL-0401 on Expressionof HBV HBsAg in Transgenic Mice

1. Experimental Protocol

The experimental assay was performed on male HBV transgenic mice ofproper age (requiring that HBsAg was significantly expressed). 10 miceweighing about 25 g were chosen and randomly divided into 2 groups, acontrol group and an administration group respectively, with 5 mice ineach group. On Day 0, each mouse was administered at 3 mg/kg bysubcutaneous injection with an administration volume of 100-200 μL.Before administration, blood was taken to determine HBsAg, and the levelof HBsAg in various groups was tried to be kept consistent. Whole bloodwas collected from orbital venous plexus of mice at the following timepoints: before administration (Day 0), after administration-Week 1, Week2, Week 3, Week 4, Week 5 and Week 6, to detect HBsAg and investigatethe persistence of GBL-0401 in inhibiting the expression of HBV gene.

The specific administration information was shown in the table below:

Administration Number of Administration No. Test drug dosage mice/groupSolvent route 1 Blank — 5 Normal Subcutaneous solvent saline injection 2GBL-0401 3 mg/kg 5 Normal Subcutaneous saline injection

2. Samples and Reagents

No. Name Specification Purity/Content 1 GBL-0401 500 μg/vial*1 vial92.3% 2 Normal saline 500 ml/bottle 0.9%

3. Kit

Kit Name Lot No. Manufacturer Kit for hepatitis B virus surface 39531900Roche Diagnostics antigen (Electrochemiluminescence) (Shanghai) Ltd. Co.

4. Experimental instruments

Name Model No. Manufacturer Vortex blender MIX-28 DragonLAB CentrifugeS1010E THERMO Full-automatic 602 Roche Diagnostics GmbH chemiluminescentanalyzer

5. Test Results

The results showed that, GBL-0401 reached the optimal inhibitory effectof 99.08% at Week 1, with a slightly decreasing trend from Week 2 toWeek 3, but still presented a high inhibitory rate of about 90%, and adeclining trend from Week 4 to Week 6, but still maintained aninhibitory effect of about 75%. GBL-0401 has a continuous inhibitoryeffect on the expression of HBV HBsAg, and can inhibit the expressionstably for a period of about 6 weeks. The diagram showing the in vivoinhibitory effect of GBL-0401 on HBV HbsAg is shown in FIG. 10 .

The invention claimed is:
 1. A compound comprising an interferingnucleic acid having a sense strand and an antisense strand and deliverychains, wherein the compound has a structure of formula (I):

wherein: R₁ has the structure of —NH(CH₂)_(x)CH₂—, wherein x is aninteger of 3-10; R₂ has the structure of —NHCH₂CH(OH)CH₂—; the moiety

has the structure of

and the moiety

has the structure of


2. A compound comprising an interfering nucleic acid having a sensestrand and an antisense strand and delivery chains, wherein the compoundhas a structure of formula (I):

wherein: R₁ has the structure of —NH(CH₂)₅CH₂—; R₂ has the structure of—NHCH₂CH(OH)CH₂—; the moiety

has the structure of

the moiety

has the structure of


3. The compound of claim 1, wherein said interfering nucleic acid issiRNA, miRNA or Agomir.
 4. The compound of claim 1, wherein saidinterfering nucleic acid is siRNA.
 5. A pharmaceutical compositioncomprising the compound of claim 1 and a pharmaceutically acceptableauxiliary material.
 6. The compound of claim 1, wherein said interferingnucleic acid is miRNA.
 7. The compound of claim 1, wherein saidinterfering nucleic acid is Agomir.
 8. A pharmaceutical compositioncomprising the compound of claim 3 and a pharmaceutically acceptableauxiliary material.
 9. A pharmaceutical composition comprising thecompound of claim 4 and a pharmaceutically acceptable auxiliarymaterial.
 10. A pharmaceutical composition comprising the compound ofclaim 6 and a pharmaceutically acceptable auxiliary material.
 11. Apharmaceutical composition comprising the compound of claim 7 and apharmaceutically acceptable auxiliary material.
 12. The compound ofclaim 2, wherein said interfering nucleic acid is siRNA, miRNA orAgomir.
 13. The compound of claim 2, wherein said interfering nucleicacid is siRNA.
 14. The compound of claim 2, wherein said interferingnucleic acid is miRNA.
 15. The compound of claim 2, wherein saidinterfering nucleic acid is Agomir.
 16. A pharmaceutical compositioncomprising the compound of claim 2 and a pharmaceutically acceptableauxiliary material.
 17. A pharmaceutical composition comprising thecompound of claim 12 and a pharmaceutically acceptable auxiliarymaterial.
 18. A pharmaceutical composition comprising the compound ofclaim 13 and a pharmaceutically acceptable auxiliary material.
 19. Apharmaceutical composition comprising the compound of claim 14 and apharmaceutically acceptable auxiliary material.
 20. A pharmaceuticalcomposition comprising the compound of claim 15 and a pharmaceuticallyacceptable auxiliary material.